Joseph Manetta

Regulation of Expression of ob mRNA and Protein by Glucocorticoids and cAMP

Regulation of obese gene (ob) expression in ob/ob and db/db mice and in cultured rat adipocytes was examined. It has been demonstrated that exogenous human OB protein (leptin) treatment reduces food intake and weight gain, as well as insulin, glucose, and corticosterone levels in ob/ob mice. In the present report we show that leptin treatment down-regulates endogenous adipose ob mRNA. However, treatment of isolated rat adipocytes with 100 ng/ml human or murine leptin had no direct effect on expression of endogenous ob mRNA, suggesting that leptin may be able to down-regulate its own expression by an indirect, non-autocrine mechanism. Glucocorticoids increased both ob mRNA levels and secreted leptin levels in vitro. Conversely, agents that increase intracellular cAMP, such as β-adrenergic agonists or BtcAMP itself, decreased ob mRNA expression and leptin secretion. Therefore, increased glucocorticoid levels and decreased sympathetic neural activity may contribute to the elevated ob mRNA expression observed in genetically obese, hyperglucocorticoid rodents. Furthermore, leptin might regulate its own expression through a feedback mechanism involving the hypothalamic pituitary axis.

Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is expressed in human brain astrocytes

Calcium-sensitive cytosolic phospholipase A2 (cPLA2) is responsible for receptor-mediated liberation of arachidonic acid, and thus plays an important role in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. In this study we have investigated the cellular distribution of cPLA2 in brain using a monoclonal antibody raised against cPLA2 to immunostain tissue sections of human cerebral cortex. We have localized cPLA2 in astrocytes of the gray matter. Colocalization with glial fibrillary acidic protein (GFAP) confirmed that cPLA2 is associated predominantly with protoplasmic astrocytes. Astrocytes of the white matter, on the other hand, were not immunoreactive. In experiments using different human astrocytoma cell lines we found that cPLA2 can be immunochemically localized in UC-11 MG cells, but cannot be detected in U-373 MG cells. This finding is consistent with the observation that cPLA2 mRNA as well as cPLA2 enzymatic activity can be readily measured in UC-11 MG astrocytoma cells, yet cannot be detected in U-373 MG cells. Our data suggest that the astrocyte is a primary source of cPLA2 in the brain and provide further evidence for the importance of this cell type in inflammatory processes in the brain.

Glucose Transporter Levels in Tissues of Spontaneously Diabetic Zucker fa/fa Rat (ZDF/drt) and Viable Yellow Mouse (Avy/a)

We used antibodies to the fat/muscle glucose transporter (GLUT4) and the liver glucose transporter (GLUT2) to measure levels of these proteins in various tissues of two rodent models of non-insulin-dependent (type II) diabetes mellitus: the obese spontaneously diabetic male Zucker fa/fa rat (ZDF/drt) and the male viable yellow Avy/a obese diabetic mouse. The ZDF/drt strain generally develops overt diabetes associated with decreased plasma insulin levels. Depending on the age of the animals, the ZDF/drt rats can be arbitrarily segregated into age-matched obese, mildly diabetic (blood glucose < 11 mM) and obese, and severely diabetic (blood glucose > 20 mM) groups. Avy/a mice are comparably hyperglycemic but unlike the ZDF/drt rats are severely hyperinsulinemic. In both groups of diabetic animals, GLUT4 in adipose tissue, heart, and skeletal muscle was reduced 25–55%, and GLUT2 in liver was increased 30–40%, relative to lean, age-matched controls. However, when the mildly diabetic ZDF/drt rats were compared to the lean controls, the only significant difference was a 25% reduction of GLUT4 in heart. Within all of the ZDF/drt rats (excluding the lean controls), GLUT2 in liver and GLUT4 in adipose tissue, heart, and skeletal muscle correlated significantly with glycemia. These data suggest that, in these two models of type II diabetes, glucose transporter levels in muscle, adipose tissue, and liver are regulated in a tissue-selective manner in response to changes in insulin and glucose. Furthermore, at least in the ZDF/drt rat, alterations in GLUT2 and/or GLUT4 protein levels appear not to be associated with obesity per se but appear to be secondary to the severely diabetic state. In both rodent models, similar alterations in GLUT2 and/or GLUT4 levels were observed under comparable glycemia, but quite distinct insulin levels, consistent with the possibility that glucose itself may be an important contributing regulator of glucose transporter expression.

Acylation of Human Insulin With Palmitic Acid Extends the Time Action of Human Insulin in Diabetic Dogs

To test whether the binding of insulin to an endogenous serum protein can be used to extend the time action of insulin, human insulin was acylated at the epsilonamino group of Lys(B29) with palmitic acid to promote binding to serum albumin. Size-exclusion chromatography was used to demonstrate specific binding of the resulting analog, [N∈-palmitoyl Lys(B29)] human insulin, to serum albumin in vitro, and the time action and activity of the analog were determined in vivo using overnight-fasted, insulin-withdrawn diabetic dogs. In the diabetic animal model, the duration of action of [N∈-palmitoyl Lys(B29)] human insulin administered intravenously was nearly twice that of unmodified human insulin, and the plasma half-life was nearly sevenfold that of the unmodified protein. Administered subcutaneously, [N∈-palmitoyl Lys(B29)] human insulin had a longer duration of action; a flatter more basal plasma insulin profile; and a lower intersubject variability of response than the intermediate-acting insulin suspension Humulin L (Lilly, Indianapolis, IN). These studies support the concept that modification of insulin to promote binding to an existing serum protein can be used to extend the time action of human insulin. In addition, the time action, pattern, and decreased variability of response to [N∈-palmitoyl Lys(B29)] human insulin support the development and further testing of this soluble insulin analog as a basal insulin to increase the safety of intensive insulin therapy.

A Dual-Monoclonal Sandwich ELISA Specific for Hepcidin-25

BACKGROUND Hepcidin, a key regulator of iron metabolism, binds to the iron transporter ferroportin to cause its degradation. In humans, hepcidin deficiency has been linked to hemochromatosis and iron overload, whereas increased concentrations have been reported in anemia of cancer and chronic disease. There is currently an unmet clinical need for a specific immunoassay with a low limit of quantification to measure serum concentrations of hepcidin-25, the active form of the protein. METHODS We generated 2 antihepcidin-25 monoclonal antibodies and used them to build a sandwich ELISA. We correlated ELISA results to hepcidin-25 measurements by LC-MS and used ELISA to measure serum hepcidin-25 concentrations in normal individuals, cancer patients, and patients with rheumatoid arthritis. RESULTS The sandwich ELISA was highly specific for hepcidin-25, having a limit of quantification of 0.01 μg/L (10 pg/mL). Serum concentrations of hepcidin-25 measured by ELISA correlated with hepcidin-25 concentrations measured by using an independent LC-MS assay (r = 0.98, P < 0.001). Hepcidin-25 concentrations were increased in patients with cancer (median 54.8 μg/L, 25%-75% range 23.2-93.5 μg/L, n = 34) and rheumatoid arthritis (median 10.6 μg/L, 25%-75% range 5.9-18.4 μg/L, n = 76) compared with healthy individuals (median 1.20 μg/L, 25%-75% range 0.42-3.07 μg/L, n = 100). CONCLUSIONS The use of 2 monoclonal antibodies in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of the active form of hepcidin. This ELISA should help to improve our understanding of the role of hepcidin in regulating iron metabolism.

Generation and characterization of tabalumab, a human monoclonal antibody that neutralizes both soluble and membrane-bound B-cell activating factor.

B-cell activating factor (BAFF) is a B-cell survival factor with a key role in B-cell homeostasis and tolerance. Dysregulated BAFF expression may contribute to autoimmune diseases or B-cell malignancies via effects on abnormal B-lymphocyte activation, proliferation, survival, and immunoglobulin secretion. Monoclonal antibodies were generated against human BAFF, characterized for species specificity and affinity, and screened for the ability to neutralize both membrane-bound and soluble BAFF. In addition, studies were undertaken to determine the relative potency of membrane-bound and soluble BAFF. Tabalumab has a high affinity for human, cynomolgus monkey, and rabbit BAFF. No binding to mouse BAFF was detected. Tabalumab was able to neutralize soluble human, cynomolgus monkey, or rabbit BAFF with equal potency. Our data demonstrate that membrane-bound BAFF can be a more potent stimulus for B-cells than soluble BAFF, and tabalumab also neutralized membrane-bound BAFF. Tabalumab prevented BAFF from binding to BAFF receptors and demonstrated pharmacodynamic effects in human BAFF transgenic mice. Tabalumab is a high-affinity human antibody with neutralizing activity against membrane-bound and soluble BAFF. Given our findings that membrane-bound BAFF can have greater in vitro potency than soluble BAFF, neutralization of both forms of BAFF is likely to be important for optimal therapeutic effect.

Design and implementation of a particle concentration fluorescence method for the detection of HIV-1 protease inhibitors.

A critical step in the replicative cycle of the human immunodeficiency virus HIV-1 involves the proteolytic processing of the polyprotein products Prgag and Prgag-pol that are encoded by the gag and pol genes in the viral genome. Inhibitors of this processing step have the potential to be important therapeutic agents in the management of acquired immunodeficiency syndrome. Current assays for inhibitors of HIV-1 protease are slow, cumbersome, or susceptible to interference by test compounds. An approach to the generation of a rapid, sensitive assay for HIV-1 protease inhibitors that is devoid of interference problems is to use a capture system which allows for isolation of the products from the reaction mixture prior to signal quantitation. In this paper, we describe a novel method for the detection of HIV-1 protease inhibitors utilizing the concept of particle concentration fluorescence. Our approach involves the use of the HIV-1 protease peptide substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val which has been modified to contain a biotin moiety on one side and a fluorescein reporter molecule on the other side of the scissile Tyr-Pro bond. This substrate is efficiently cleaved by the HIV-1 protease and the reaction can be readily quantitated. Known inhibitors of the protease were readily detected using this new assay. In addition, this approach is compatible with existing instrumentation in use for broad screening and is highly sensitive, accurate, and reproducible.

Maternal Serum B-Cell Activating Factor Levels: Candidate Early Biomarker for Hypertensive Disorders of Pregnancy

Hypertensive disorders of pregnancy are a leading cause of maternal and perinatal morbidity and mortality. Early suppression of B-cell lymphopoiesis is necessary for a normal pregnancy. Dysregulation of factors critical to B-cell survival may result in pregnancy complications, including hypertension. In this prospective observational study at a single medical center, serum levels of BAFF (B-cell activating factor) were measured in pregnant participants at each trimester, at delivery, and postpartum and in nonpregnant controls at a single time point. Comparisons were made between nonpregnant and pregnant subjects and between time periods of pregnancy. First-trimester serum BAFF levels were further tested for association with hypertensive disorders of pregnancy. The study included 149 healthy pregnant women, 25 pregnant women with chronic hypertension, and 48 nonpregnant controls. Median first-trimester serum BAFF level (ng/mL) for healthy women (0.90) was lower than median serum BAFF levels for women with chronic hypertension (0.96; P=0.013) and controls (1.00; P=0.002). Serum BAFF levels steadily declined throughout pregnancy, with the median second-trimester level lower than the corresponding first-trimester level (0.77; P=0.003) and the median third-trimester level lower than the corresponding second-trimester level (0.72; P=0.025). The median first-trimester serum BAFF level was elevated in women who subsequently developed hypertension compared with women who remained normotensive (1.02 versus 0.85; P=0.012), with the area under the receiver operating characteristic curve being 0.709. First-trimester serum BAFF level may be an early and clinically useful predictor of hypertensive disorders of pregnancy.